Hypoxanthine

The P. falciparum strains 3D7 and K1 were cultivated in complete medium consisting of the following components: Roswell Park Memorial Institute (RPMI)-1640 (Sigma‒Aldrich, St. Louis, MO, USA), 25 mM HEPES (CAS: 7365-45-9, Sigma‒Aldrich, St. Louis, MO, USA), 0.5% (w/v) AlbuMax™ II (lipid-rich BSA, Gibco, Waltham, MA, USA), 24 mM NaHCO₃ (CAS: 144-55-8, Wako, Osaka, Japan), 184 μM hypoxanthine (CAS: 68-94-0, Wako, Osaka, Japan), and 0.025% (v/v) gentamicin (50 mg/mL, CAS: 1403-66-3, Gibco, Waltham, MA, USA) supplemented with 2% washed human O-(+) erythrocytes, provided by the Japanese Red Cross Society, Hokkaido, Japan. The cultures were maintained in an incubator under specific conditions: 37 °C under 5% CO₂ and 5% O₂. The parasitemia levels were monitored using Giemsa-stained thin blood smears (CAS: 51811-82-6, Merck, Darmstadt, Hesse, Germany). Ethical approval for human blood malaria parasite culture was obtained with permit number #2013-04-3 from the ethical committee of Obihiro University of Agriculture and Veterinary Medicine adhering to the ethical standards for research involving human blood samples.

[³H]Adenine (25.0 Ci/mmol), [³H]hypoxanthine (27.0 Ci/mmol), [³H]thymidine (20.0 Ci/mmol), [³H]uridine (14.7 Ci/mmol), [³H]uracil (42.8 Ci/mmol), [³H]xanthine (12.8 Ci/mmol) and [³H]adenosine (39.2 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA), [¹⁴C]guanine (55 mCi/mmol) was from American Radiolabeled Chemicals (St. Louis, MO), and [¹⁴C]ascorbate (8.5 mCi/mmol) was from PerkinElmer Life Sciences (Boston, MA). Adenine, papaverine, and dipyridamole were obtained from Sigma-Aldrich (St. Louis, MO), and hypoxanthine, guanine and nitrobenzylthioinosine were from Wako Pure Chemicals (Osaka, Japan). All other reagents were of analytical grade and commercially obtained.

HEK293T cells were cultured in Dulbecco's modified Eagle's Medium (D6546; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (S1820-500; Biowest/173012; Sigma-Aldrich), 50 U/ml penicillin, 50 μg/ml streptomycin (15070-063; GIBCO), and 2 mM glutamine (25030-081; GIBCO) in a 5% CO₂ incubator (hereafter referred to as normal culture conditions). For purine depletion experiments, cells were cultured in purine-depleted media, which is Dulbecco's modified Eagle's Medium supplemented with 10% dialyzed FBS (SH30079; Hyclone), 50 U/ml penicillin, 50 μg/ml streptomycin, and 2 mM glutamine. In the indicated experiments, cells were cultured with 2 μM methotrexate (135-13573; Wako), 10 μg/ml pepstatin A (4397-v; Peptide Institute), 10 μg/ml E64d (4321-v; Peptide Institute), hypoxanthine (086-03403; Wako), adenine (A0149; Tokyo Chemical Industry), guanine (G0169; Tokyo Chemical Industry), inosine (I4125; Sigma-Aldrich), or inosine 5'-monophosphate disodium salt hydrate (I0036; Tokyo Chemical Industry). Except for the screen, cells lacking de novo purine synthesis were maintained in media supplemented with 100 μM hypoxanthine, and the media were changed before analysis.

B. breve MCC1274 was cultured in YC medium supplemented with 200 μM each of adenosine, inosine, hypoxanthine, and xanthine (FUJIFILM Wako Pure Chemical) for 24 h under anaerobic conditions. Then, the culture medium was centrifuged (10,000 × g, 5 min, 4°C), and the supernatant was stored at −20°C until analysis.

Reagents were obtained from the following suppliers: hydrogen peroxide 30%, iron (II) sulfate heptahydrate, NaClO, and hypoxanthine that were from Wako Pure Chemical Industries Ltd. (Osaka Japan). Xanthine oxidase was obtained from bovine milk and pyrrolidine from SIGMA-ALDRICH, Co (USA). Diethylenetriaminepentaacetic acid (DETAPAC) and NOC-7 were obtained from DOJIN MOLECULAR TECHNOLOGIES, INC. All other chemicals were obtained as reagent-grade.
Probe 1 was donated kindly from Professor K. Koide of University of Pittsburgh [6 ].
Ozone-containing water was prepared by the electrolysis of water (ozone generator: VMC JAPAN). The concentration of standard ozone-containing water was calculated from its absorbance at a wavelength of 254 nm using a molar absorptivity coefficient, ε, of 2950 mol⁻¹Lcm⁻¹ [7 ].
Preparation of probe 1 solution for assay: a solution of probe 1 (12.5 μmol/L) in a mixture of 5% v/v methanol in a 0.5 mmol/L phosphate buffer (pH 3.3) was prepared immediately prior to use.